Receptor tyrosine kinases play a diverse role in cell growth and differentiation during normal physiologic responses and in oncogenic transformation and tumor progression. Eph receptors are a unique family of receptor tyrosine kinases (RTK), the largest in the genome, consisting of at least 16 receptors that interact with nine membrane-bound ephrin ligands (Pasquale, E. B. et al., 2005, Nature Reviews Mol. Cell. Biol., 6: 462-475). They can be further divided into two groups, class A and B, based on the sequence homology and binding affinity (Pasquale, E. B. et al., 2005, Nature Reviews Mol. Cell. Biol., 6: 462-475). Class A Eph receptors interact with multiple ligands of the ephrin-A family, a group of glycosyl-phosphatidylinositol (GPI)-linked membrane proteins, while class B Eph receptors bind to ephrin-B ligands, a family of transmembrane proteins. Binding of Eph receptors to their ligands induces receptor clustering, activation of kinase activity, and subsequent trans-phosphorylation of the cytoplasmic domains on tyrosine residues, creating docking sites for a number of signaling proteins (Kullander, K. and Klein, R., 2002, Nature Reviews Mol. Cell. Biol., 3: 475-486; Noren, N. K. and Pasquale, E. B., 2004, Cell signal., 16: 655-666).
Cancer is a disease characterized by uncontrolled proliferation, resulting from aberrant signal transduction. The most dangerous forms of cancer are malignant cells which have the ability of these to spread, either by direct growth into adjacent tissue through invasion, or by implantation into distant sites by metastasis. Metastatic cells have acquired the ability to break away from the primary tumor, translocate to distant sites through the bloodstream or lymphatic system, and colonize distant and foreign microenvironments.
It is now clear that the Eph molecules also have a role in disease states such as cancer. In particular, overexpression of the EphA2 receptor has been reported in cancers of the ovary, breast, prostate, lung, colon, oesophagus, renal cell, cervix, and melanoma. EphA2 was suggested to be a positive regulator of cell growth and survival in malignant cells (Landen, C. N. et al., 2005, Expert. Opin. Ther. Targets, 9 (6): 1179-1187). A role for EphA2 in metastasis has also been described, since EphA2 overexpression alone is sufficient to transform mammary epithelial cells into a malignant phenotype (Zelinski et al., 2001, Cancer Res., 61: 2301-2306), and increases spontaneous metastasis to distant sites (Landen, C. N. et al., 2005, Expert. Opin. Ther. Targets, 9 (6): 1179-1187). Furthermore, increasing evidence suggests that EphA2 is involved in tumor angiogenesis (Ogawa et al., 2000, Oncogene, 19: 6043-6052; Cheng et al. 2002, Mol. Cancer. Res., 1: 2-11; Cheng et al., 2003, Neoplasia, 5 (5): 445-456; Dobrzanski et al., 2004, Cancer Res., 64: 910-919).
Phosphorylation of EphA2 has been shown to be linked to its abundance. Tyrosine phosphorylated EphA2 is rapidly internalised and fated for degradation, whereas unphosphorylated EphA2 demonstrates reduced turnover and therefore accumulates at the cell surface. It is currently thought that this kind of model might contribute to the high frequency of EphA2 overexpression in cancer (Landen, C. N. et al., 2005, Expert. Opin. Ther. Targets, 9 (6): 1179-1187). However, reality may be more complex, since recent data seem to indicate a role for EphA2 kinase-dependent and -independent functions in tumor progression (Fang W. B., 2005, Oncogene, 24: 7859-7868).
Agonistic antibodies have been developped which promote EphA2 tyrosine phosphorylation and internalisation, ultimately resulting in inhibition of tumor cell growth (Dodge-Zantek et al., 1999, Cell Growth & Differ., 10: 629-638; WO 01/12172, WO 03/094859, WO 2004/014292, WO 2004/101764, WO 2006/023403, WO 2006/047637, WO 2007/030642). These antibodies are directed against the extracellular domain of EphA2. Since these agonist antibodies do not inhibit but rather stimulate EphA2 receptor phosphorylation and downstream signals, these antibodies might not be effective for tumors which take advantage of the EphA2 kinase activity. On the other hand, the use of antagonistic agents, including antibodies, has been proposed (WO 2004/092343), but no actual antagonistic antibody was disclosed therein. Moreover, such antibodies were proposed to stimulate, rather than inhibit, cell proliferation. Application WO 2006/084226 discloses antibodies which neither increase nor decrease EphA2 kinase activity but are capable of impeding tumor cell proliferation. However, there is no indication therein that these antibodies prevent ephrinA1 binding to the receptor and inhibit ephrinA1-induced EphA2 phosphorylation. Rather, they may affect tumor cell proliferation through a totally different mechanism, e.g. by preventing receptor clustering following ephrinA1 binding. The skilled person would thus not have concluded that these antibodies are antagonists, but, rather, that their mechanism of action is unclear.
Therefore, there is a need for new, antagonistic anti-EphA2 antibodies, which bind to the extracellular domains of EphA2 receptor, inhibit its activation by the ligand ephrin A1 and inhibit EphA2 kinase-dependend tumor cell growth. Such antagonistic antibodies should be useful for the treatment of cancer.